Method for producing retinoid from microorganism

ABSTRACT

The present invention relates to a method for producing retinoid from a microorganism, and more specifically, to a method for effectively obtaining retinoid, which lacks stability, from a microorganism by cultivating the microorganism capable of producing retinoid in a medium containing a lipophilic substance, and separating retinoid from the lipophilic substance.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. §371 ofInternational Application No. PCT/KR2012/006071, filed Jul. 30, 2012,which claims priority to and the benefit of Republic of Korea PatentApplication Nos. 10-2011-0075715 filed Jul. 29, 2011 and 10-2012-0083185filed Jul. 30, 2012, the disclosures of which are incorporated herein byreference in its entirety.

BACKGROUND

1. Technical Field

The present invention relates to a method for production of retinoidfrom a microorganism with retinoid producing efficacy.

2. Description of the Related Art

Retinoid belongs to a class of lipophilic isoprenoid moleculeschemically associated with vitamin A. The retinoid includes β-inonecyclic and polyunsaturated branched chains, together with alcohol (e.g.,retinol), aldehyde (e.g., retinal), carboxylic acid (e.g., retinoicacid) or ester (e.g., retinyl acetate) functional groups. It is wellknown that such functional groups play an essential role in health careof a human body such as eyesight protection, bone growth andregeneration, anti-oxidation effects and prevention of skin aging, andmay also reduce danger of cancer.

In recent years, the retinoid has attracted a great interest as anefficient raw material for cosmetics and medicaments useful for wrinkleimprovement and treatment of skin disease. A scale of retinoid market isestimated at about 16 billion dollars over the world. Chemicallysynthesized retinoid is a representative raw material commerciallyavailable in the market. Retinol is produced by acidification orhydrolysis of retinal which was chemically synthesized through reductionof a pentadiene derivative. However, such a chemical process asdescribed above entails disadvantages such as complicated purificationprocesses and generation of undesirable byproducts. An animal createsretinoid using carotinoid obtained from fruits and vegetables, while aplant cannot synthesize retinoid. An overall path of retinoid synthesisis possibly embodied only in a specific microorganism that containsbacteriorhodopsin or proteorhodopsin having retinal as a prostheticgroup. Nevertheless, the microorganism generates a protein combinationform of retinal, thus not being preferable in mass production of freeretinoids. Until now, although partially restricted attempts usingenzymes for biological production have been executed, these have not yetgained successful results. Accordingly, there is still a need fordevelopment of a biotechnological method using metabolically transformedmicroorganisms in order to produce retinoid.

The retinoid is chemically unstable due to a conjugated and activedouble bond, and easily oxidized and becomes isomeric by heat, oxygenand light. Further, the retinoid is liable to be degraded throughretinoic acid in biological aspects. Accordingly, an effective methodfor production of retinoid has yet to be developed.

SUMMARY

Therefore, an object of the present invention is to provide a method foreffectively producing retinoid from a microorganism.

According to an aspect of the present invention, there is provided amethod for production of retinoid from a microorganism, including:culturing a microorganism having retinoid producing efficacy in a mediumcontaining a lipophilic substance; and isolating retinoid from thelipophilic substance phase.

The method may include culturing the microorganism having retinoidproducing efficacy in a medium containing a lipophilic substance.

The term “microorganism” used herein may include a cell possiblecultured in a liquid medium. The microorganism may include prokaryotecells, eukaryote cells or isolated animal cells, which are possiblycultured in a liquid medium. Such microorganisms may include, forexample, bacteria, fungi or a combination thereof. Bacteria may include,for example, gram positive bacteria, gram negative bacteria or acombination thereof. The gram negative bacteria may include species ofthe genus Escherichia. The gram positive bacteria may include species ofthe genus bacillus, genus corynebacterium, lactobacillus or acombination thereof. The fungi may include yeast, kluyveromyces or acombination thereof. The microorganism may have natural or foreign genesintroduced therein. The foreign gene may be a gene in association withproduction of retinoid such as at least one gene in an MEP or MVA path.The animal cell may include cells used for production of recombinantproteins. For instance, CHO cell, BHK cell or a combination thereof maybe included.

The microorganism of the genus Escherichia having retinoid producingefficacy may include microorganisms of the genus of natural ortransformed Escherichia. The microorganism in the natural genusEscherichia is known to have the MEP path as an inherent path forretinoid synthesis. The microorganism in the transformed genusEscherichia may include genes associated with an inherent MEP path forretinoid synthesis, genes associated with a foreign MVA path forretinoid synthesis or a combination thereof, which are introducedtherein. The MVA path gene may be a gene encoding an enzyme in a foreignmevalonate path in association with production of IPP from acetyl-CoA.Alternatively, a strain into which a gene encoding an enzyme associatedwith synthesis of β-carotene from the above IPP has been introduced mayalso be included. Further, the foregoing microorganism may be one havingat least two copies of IPP isomerases introduced therein, which in turn,shows promoted conversion from IPP to DMAPP. Therefore, themicroorganism may the retinoid at a high concentration. FIG. 1 is a viewschematically illustrating an MEP path and a foreign MVA path of retinalbiosynthesis.

The microorganism in the natural genus Escherichia may include, forexample, Escherichia coli. Such genus Escherichia coli may include, forexample, DH5α, MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113 or acombination thereof.

The microorganism in the transformed genus Escherichia may be onetransformed into, for example: a gene encoding acetyl-CoA acetyltransferase/hydroxymethylglutaryl(HMG)-CoA reductase derived fromEnterococcus faecalis, which is defined by SEQ. ID No. 1; a geneencoding HMG-CoA synthase derived from Enterococcus faecalis, which isdefined by SEQ. ID No. 2; a gene encoding mevalonate kinase derived fromStreptococcus pneumoniae, which is defined by SEQ. ID No. 3; a geneencoding phosphomevalonate kinase derived from Streptococcus pneumoniae,which is defined by SEQ. ID No. 4; a gene encoding mevalonatediphosphate decarboxylase derived from Streptococcus pneumoniae, whichis defined by SEQ. ID No. 5; a gene encoding isopentinyl diphosphate(IPP) isomerase derived from Escherichia coli, which is defined by SEQ.ID No. 6; a gene encoding geranylgeranyl pyrophosphate (GGPP) synthasederived from Pantoea agglomerans, which is defined by SEQ. ID No. 7; agene encoding phytoene synthase derived from Pantoea agglomerans, whichis defined by SEQ. ID No. 8; a gene encoding phytoene dehydrogenasederived from Pantoea agglomerans, which is defined by SEQ. ID No. 9; anda gene encoding lycopene β-cyclase derived from Pantoea ananatis, whichis defined by SEQ. ID No. 10.

The microorganism in the transformed genus Escherichia may be onetransformed into any one of the genes defined by SEQ. ID Nos. 1 to 10,and may be further transformed into at least one selected from a groupconsisting of, for example: a gene encoding β-carotene monooxygenasederived from uncultured marine bacterium 66A03, which is defined by SEQ.ID No. 13; a gene encoding β-carotene 15,15′-monooxygenase derived fromMus musculus, which is defined by SEQ. ID No. 14; a gene encodingbrp-like protein 2 (brp 2) derived from Natronomonas pharaonisATCC35678, which is defined by SEQ. ID No. 15; and a gene encodingβ-carotene monooxygenase derived from Halobacterium salinarumATCC700922, which is defined by SEQ. ID No. 16 or 17, or the like.

The microorganism described above may produce retinoid furthertransformed into a gene encoding IPP isomerase derived fromHaematococcus pluvialis, which is defined by SEQ. ID No. 12.

The microorganism having retinoid producing efficacy may be transformedinto a gene encoding 1-deoxyxylolose-5-phosphate (DXP) synthase (dxs)derived from Escherichia coli, which is defined by SEQ. ID No. 11. SinceDXP is an enzyme corresponding to a process of determining a velocity inan inherent MEP path, the above microorganism can produce β-carotene ata high concentration by further introducing a gene encoding DXPsynthase.

If the microorganism having retionid producing efficacy of the presentinvention belongs to the genus Escherichia, it may be, for example,Escherichia coli DH5α/pTDHB/pSNA with Accession No. KCTC 11254BP (Koreancollection for type culture, deposited on Jan. 2, 2008) or Escherichiacoli DH5α/pTDHBSR/pSNA with Accession No. KCTC 11255BP (Koreancollection for type culture, deposited on Jan. 2, 2008). In particular,or Escherichia coli DH5α/pTDHBSR/pSNA can produce retinoid with highproductivity from a carbon source in a medium. The microorganisms,Escherichia coli DH5α/pTDHB/pSNA and Escherichia coli DH5α/pTDHBSR/pSNA,were duly deposited with Korean Collection for Type Cultures (KCTC)(having the address of Biological Resource Center (BRC), Korea ResearchInstitute of Bioscience and Biotechnology (KRIBB), 52 Eoeun-dong,Yuseong-gu, Daejeon 305-333, Republic of Korea) under the Access numbersof KCTC11254BP and KCTC 11255BP, respectively, on Jan. 2, 2008. Thedeposits have been made under the terms of the Budapest Treaty and allrestrictions imposed by the depositor on the availability to the publicof the biological material will be irrevocably removed upon the grantingof a patent.

According to one embodiment, the microorganism described above may be amicroorganism in the genus Escherichia transformed into, for example: agene encoding acetyl-CoA acetyltransferase/hydroxymethylglutaryl(HMG)-CoA reductase derived from Enterococcus faecalis, which is definedby SEQ. ID No. 1; a gene encoding HMG-CoA synthase derived fromEnterococcus faecalis, which is defined by SEQ. ID No. 2; a geneencoding mevalonate kinase derived from Streptococcus pneumoniae, whichis defined by SEQ. ID No. 3; a gene encoding phosphomevalonate kinasederived from Streptococcus pneumoniae, which is defined by SEQ. ID No.4; a gene encoding mevalonate diphosphate decarboxylase derived fromStreptococcus pneumoniae, which is defined by SEQ. ID No. 5; a geneencoding isopentinyl diphosphate (IPP) isomerase derived fromEscherichia coli, which is defined by SEQ. ID No. 6; a gene encodinggeranylgeranyl pyrophosphate (GGPP) synthase derived from Pantoeaagglomerans, which is defined by SEQ. ID No. 7; a gene encoding phytoenesynthase derived from Pantoea agglomerans, which is defined by SEQ. IDNo. 8; a gene encoding phytoene dehydrogenase derived from Pantoeaagglomerans, which is defined by SEQ. ID No. 9; a gene encoding lycopeneβ-cyclase derived from Pantoea ananatis, which is defined by SEQ. ID No.10; a gene encoding 1-deoxyxylolose-5-phosphate (DXP) synthase (dxs)derived from Escherichia coli, which is defined by SEQ. ID No. 11; and agene encoding IPP isomerase derived from Haematococcus pluvialis, whichis defined by SEQ. ID No. 12. More particularly, such a microorganism inthe genus Escherichia as described above may be a microorganism in thegenus Escherichia further transformed into at least one gene selectedfrom a group consisting of, for example: a gene encoding β-carotenemonooxygenase derived from uncultured marine bacterium 66A03, which isdefined by SEQ. ID No. 13; a gene encoding β-carotene15,15′-monooxygenase derived from Mus musculus, which is defined by SEQ.ID No. 14; a gene encoding brp-like protein 2 (brp 2) derived fromNatronomonas pharaonis ATCC35678, which is defined by SEQ. ID No. 15;and a gene encoding β-carotene monooxygenase derived from Halobacteriumsalinarum ATCC70922, which are defined by SEQ. ID Nos. 16 and 17. Thegene encoding β-carotene monooxygenase derived from uncultured marinebacterium 66A03, which is defined by SEQ. ID No. 13, may have a basesequence defined by SEQ. ID No. 32, which is codon-optimized inEscherichia coli.

In the present text, the term “retinoids” means a species of chemicalsubstances chemically associated with vitamin A. The retinoid may have astructure consisting of a cyclic end group, polyene branched chain andpolar end group. A conjugate system formed by alternately aligning C═Cdouble bonds in the polyene branched chain may express color of theretinoid (usually yellow, orange or red color). Most of retinoids arechromophore. By altering the branched chain and end groups, a variety ofretinoids may be produced. Such retinoids may include retinal, retinol,retinoic acid, retinyl acetate or a combination thereof. Further, theretinoid may include any product of in vivo degradation of retinal,retinol, retinoic acid, retinyl acetate or a combination thereof.

The retinoid is a material having 20 basic carbon atoms and, accordingto fatty acid prosthetic groups bonded thereto, the final number ofcarbon atoms may be changed. For instance, in case of acetate bonding,the final number of carbon atoms is 22. On the other hand, for oleicacid bonding, the final number of carbon atoms may be 38.

The lipophilic substance described above may be an organic compoundhaving 8 to 50 carbon atoms with lipophilic properties.

The lipophilic substance may include, for example, an alkane compoundhaving 8 to 50 carbon atoms, a compound represented by Formula 1 below,a compound represented by Formula 2 below, or a combination thereof.

R₁(CO)OR₂  [Formula 1]

(wherein R₁ and R₂ are each independently alkyl having 8 to 50 carbonatoms, and CO represents a carbonyl group).

(wherein R₃, R₄ and R₅ are each independently alkyl having 8 to 50carbon atoms, and CO represents a carbonyl group).

The alkane compound having 8 to 50 carbon atoms may be straight alkane,brached alkane, cyclic alkane or a combination thereof. The alkanecompound may include compounds having carbon atoms in a range of, forexample: 8 to 46; 8 to 40; 8 to 36; 8 to 30; 8 to 26; 8 to 20; 8 to 16;8 to 12; 8 to 10; 10 to 50; 10 to 46; 10 to 40; 10 to 36; 10 to 30; 10to 26; 10 to 20; 10 to 17; to 12; 10 to 50; 10 to 46; 12 to 50; 12 to46; 12 to 36; 12 to 30; 12 to 26; 12 to 20; or 12 to 16.

The straight alkane may include alkanes having 8 carbon atoms (octane),10 carbon atoms (decane), 12 carbon atoms (dodecane), 14 carbon atoms(tetradecane), or alkanes having 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48 or 50 carbon atoms, or a combination thereof.

The branched alkane may include alkanes having 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 carbonatoms, or a combination thereof. The branched alkane may includesaturated analogues of a terpene compound. For instance, phytosqualanemay be included.

A combination of the straight alkane, branched alkane and cyclic alkanemay be mineral oil. The mineral oil may be a mixture of alkanes having15 to 40 carbon atoms, which are derived from non-vegetable rawmaterials (mineral). The alkane having 15 to 40 carbon atoms mayinclude, for example, at least two mixtures of alkanes having 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 carbon atoms.

The mineral oil may be lightweight or heavy mineral oil. The lightweightmineral oil is a substance generally having a density of 880 to 920kg/m³, a specific gravity of 820 to 860 kg/m³ at 20° C., and a fluidviscosity of 14 to 18 cst at 40° C. On the other hand, the heavy mineraloil is a substance generally having a density of 920 kg/m3, a specificgravity of 860 to 900 kg/m³ at 20° C., and a fluid viscosity of 65 to 85cst at 40° C.

In the compound represented by Formula 1, R₁ and R₂ are eachindependently straight, branched or cyclic alkyl having 8 to 50 carbonatoms. Herein, R₁ and R₂ may be each independently alkyl having 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48 or 50 carbon atoms.

R₁ and R₂ may be each independently alkyl having carbon atoms in a rangeof 8 to 50, for example: 8 to 46; 8 to 40; 8 to 36; 8 to 30; 8 to 26; 8to 20; 8 to 16; 8 to 12; 8 to 10; 10 to 50; 10 to 46; 10 to 40; 10 to36; 10 to 30; 10 to 26; 10 to 20; 10 to 16; 10 to 12; 10 to 50; 10 to46; 12 to 50; 12 to 46; 12 to 36; 12 to 30; 12 to 26; 12 to 20; or 12 to16. R₁ may be a straight alkyl having 13 carbon atoms while R₂ isisopropyl. Further, R₁ may be ethylpentyl while R₂ is cetyl.

In the compound represented by Formula 2, R₃, R₄ and R₅ are eachindependently straight, branched or cyclic alkyl having 8 to 50 carbonatoms.

R₃, R₄ and R₅ may be each independently alkyl having 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50carbon atoms. The foregoing compound may contain R₃, R₄ and R₅, whichare each independently alkyl having carbon atoms in a range of 8 to 50,for example: 8 to 46; 8 to 40; 8 to 36; 8 to 30; 8 to 26; 8 to 20; 8 to16; 8 to 12; 8 to 10; 10 to 50; to 46; 10 to 40; 10 to 36; 10 to 30; 10to 26; 10 to 20; 10 to 16; 10 to 12; 10 to 50; 10 to 46; 12 to 50; 12 to46; 12 to 36; 12 to 30; 12 to 26; 12 to 20; or 12 to 16.

The lipophilic substance may include, for example, octane, decane,dodecane, tetradecane, phytosqualane, mineral oil, isopropyl myristate,cetyl ethylhexanoate, dioctanoyl decanoyl glycerol, squalane, or acombination thereof.

The lipophilic substance may not only stabilize produced retinoids butalso increase productivity of the retinoids by microorganisms. Thelipophilic substance does not affect growth of microorganisms or mayslightly influence on the same.

Culture may be performed in a synthetic, semi-synthetic or combinedculture medium. Such a culture medium may include, for example, a mediumconsisting of a carbon source, nitrogen source, vitamin and mineral. Forinstance, a Man-Rogosa-Sharp (MRS) liquid medium or a milk-added liquidmedium may be used.

The carbon source of the medium may include starch, dextrose, sucrose,galactose, fructose, glycerol, glucose, or a mixture thereof. Forinstance, glycerol may be used as the carbon source. The nitrogen sourcemay include ammonium sulfate, ammonium nitrate, sodium nitrate, glutamicacid, casaminoic acid, yeast extract, peptone, tryptone, soy bean husk,or a mixture thereof. Mineral may include sodium chloride, potassium(II) phosphate, magnesium sulfate, or a mixture thereof.

When the culturing is executed in a fermentation tank, glucose ispreferably used as a carbon source. For test tube culture, glycerol ispreferably used as a carbon source.

The carbon source, nitrogen source and mineral in the medium forculturing microorganisms are used in an amount of 10 to 100 g, 5 to 40 gand 0.5 to 4 g, respectively, to 1 liter.

Vitamin added to a typical culture medium under general cultureconditions of Escherichia coli may be vitamin A, vitamin B, vitamin C,vitamin D, vitamin E or a mixture thereof. The vitamin may be added tothe typical culture medium together with such carbon source, nitrogensource and/or mineral as described above. Otherwise, the vitamin may bealternatively added to a sterilized medium.

The culturing may be executed under general culture conditions. Theculturing may be executed at a temperature in a range of 15 to 45° C.,for example, 15 to 44° C.; 15 to 43° C.; 15 to 42° C.; 15 to 41° C.; 15to 40° C.; 15 to 39° C.; 15 to 38° C.; 15 to 37° C.; 15 to 36° C.; 15 to35° C.; 15 to 34° C.; 15 to 33° C.; 15 to 32° C.; 15 to 31° C.; 15 to30° C.; 20 to 45° C.; 20 to 44° C.; 20 to 43° C.; 20 to 42° C.; 20 to41° C.; 20 to 40° C.; 20 to 39° C.; 20 to 38° C.; 20 to 37° C.; 20 to36° C.; 20 to 35° C.; 20 to 34° C.; 20 to 33° C.; 20 to 32° C.; 20 to31° C.; 20 to 30° C.; 25 to 45° C.; 25 to 44° C.; 25 to 43° C.; 25 to42° C.; 25 to 41° C.; 25 to 40° C.; 25 to 39° C.; 25 to 38° C.; 25 to37° C.; 25 to 36° C.; 25 to 35° C.; 25 to 34° C.; 25 to 33° C.; 25 to32° C.; 25 to 31° C.; 25 to 30° C.; 27 to 45° C.; 27 to 44° C.; 27 to43° C.; 27 to 42° C.; 27 to 41° C.; 27 to 40° C.; 27 to 39° C.; 27 to38° C.; 27 to 37° C.; 27 to 36° C.; 27 to 35° C.; 27 to 34° C.; 27 to33° C.; 27 to 32° C.; 27 to 31° C.; or 27 to 30° C.

In order to collect or recover concentrated strain after removing theculture medium in a culture solution, centrifugation or filtration maybe conducted. Such a process may be optionally executed according todemands of those skilled in the art. By freezing or freeze-drying theconcentrated strain according to any conventional method, activities ofthe strain may be retained.

According to one example of the culturing, the culturing may beperformed in a medium containing glycerol as a carbon source. Glycerolmay be only the carbon source in the medium. The culturing may beconducted in a medium containing glycerol in an amount of 0.5 to 5.0%(w/v), for example: 0.5 to 4.5% (w/v); 0.5 to 4.0% (w/v); 0.5 to 3.5%(w/v); 0.5 to 3.0% (w/v); 0.5 to 2.5% (w/v); 0.5 to 2.0% (w/v); 0.5 to1.5% (w/v); 1 to 4.5% (w/v); 1 to 4.0% (w/v); 1 to 3.5% (w/v); 1 to 3.0%(w/v); or 1 to 2.5% (w/v). Such a medium may be an YT medium includingglycerol and arabinose added thereto. The YT medium may include 1.6 wt.% of tryptone, 1 wt. % of yeast extract and 0.5 wt. % of NaCl.

The culturing may be performed in a culture medium in the presence of alipophilic substance while placing a dodecane phase formed of alipophilic substance on the surface of the medium. The culturing may beperformed under agitation.

The agitation may be conducted at a range of 100 to 300 rpm, forexample, 100 to 280 rpm, 100 to 260 rpm, 100 to 240 rpm, 100 to 220 rpm,100 to 200 rpm, 100 to 180 rpm, 100 to 160 rpm, 100 to 140 rpm, 100 to120 rpm, 120 to 300 rpm, 120 to 280 rpm, 120 to 260 rpm, 120 to 240 rpm,120 to 220 rpm, 120 to 200 rpm, 120 to 180 rpm, 120 to 160 rpm, 120 to140 rpm, 150 to 300 rpm, 150 to 280 rpm, 150 to 260 rpm, 150 to 240 rpm,150 to 220 rpm, 150 to 200 rpm, 150 to 180 rpm, 140 to 160 rpm, 200 to300 rpm, 200 to 280 rpm, 200 to 260 rpm, 200 to 240 rpm, 200 to 220 rpm,or 150 rpm.

In case of agitating, the lipophilic substance, that is, dodecane may bedispersed in the medium and contact with cells. Since the lipophilicsubstance is dispersed in the medium to increase amicroorganism-contacting area, retinoid may be efficiently isolated fromthe cells during culturing, thereby enabling stabilization anddissolution of the retinoid.

When a microorganism for producing retinoids was cultured without thelipophilic substance, that is, the dodecane phase, the production ofretinoids may reach a maximum level at a constant time and, thereafter,become decrease. The reason of such facts may be because a furthersynthesis of retinoid is stopped during the growth of microorganism hasstagnated, while occurring intracellular oxidative degradation of theretinoid.

If the microorganism is cultured in a culture medium in the presence ofthe lipophilic substance, that is, dodecane phase, the produced retinoidmay be absorbed in to the lipophilic substance, that is, dodecane phasebefore the retinoid is degraded in the cell, thereby improvingproductivity of retinoid.

The lipophilic substance, that is, dodecane phase does not affect uponthe cellular growth of a microorganism in the genus Escherichia,instead, may be hydrophobic and used for extracting the retinoid andhave a low volatility. FIG. 2 illustrates conversion of β-carotene intoretinoids including retinal, retinol, retinoic acid and retinyl ester.

A ratio by volume of medium to lipophilic substance may be, for example:1:0.1 to 3.0, 1:0.5 to 3.0, 1:1.0 to 3.0, 1:1.5 to 3.0, 1:2.0 to 3.0,1:2.5 to 3.0, 1:0.2 to 2.5, 1:0.2 to 2.0, 1:0.2 to 1.5, 1:0.2 to 1.0,1:0.2 to 0.5, 1:0.5 to 2.5, 1:0.5 to 2.0, 1:0.5 to 1.5, 1:0.5 to 1.0,1:0.8 to 2.5, 1:0.8 to 2.0, 1:0.8 to 1.5, 1:0.8 to 1.2, 1:0.8 to 1.0, orthe like.

According to one embodiment, the medium may contain glycerol at aconcentration of about 2.0% during culturing, the microorganism in thegenus Escherichia may be Escherichia coli DH5α or MG1655, and theculturing may include culturing using 7 ml of a culture solution atabout 29° C.

The foregoing method may include isolating retinoid from a lipophilicsubstance phase. A method for isolation of the retinoid including, forexample, retinal, retinol, retinoic acid, retinyl ester or a combinationthereof is well known in the art. For example, the retinoid may beisolated by a conventional method such as ion exchange chromatography,HPLC or the like. For instance, in order to obtain a high purity productafter recovering a strain and extracting the same using a solvent suchas acetone, separation and purification through HPLC or crylstallizationmay be conducted.

A method for production of retinoid from a microorganism in the genusEscherichia according to one embodiment of the present invention mayinclude: culturing the microorganism having retinoid producing efficacyin the genus Escherichia, in a medium including a lipophilic substance;and isolating the retinoid from the lipophilic substance phase, whereinthe lipophilic substance is an alkane compound having 8 to 50 carbonatoms, a compound represented by Formula 1, a compound represented byFormula 2 or a combination thereof.

The method for production of retinoid according to the present inventionmay produce retinol with high efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view schematically illustrating an MEP path and a foreignMVA path of retinal biosynthesis;

FIG. 2 illustrates retinoid conversion of β-carotene into retinoidincluding retinal, retinol, retinoic acid and retinyl ester.

FIG. 3 illustrates production of retinal, production of β-carotene andcell growth of Escherichia coli including pT-HB, pT-HBblh, pT-HPbrp,pT-HBbrp2, pT-HBBCMO1 and pT-HBSR;

FIG. 4 illustrates production of retinal, production of β-carotene andcell growth of Escherichia coli including pT-HB, pT-HBSR, pT-DHB andpT-DHBSR, as well as Escherichia coli including pT-DHB or pT-DHBSRtogether with pS-NA as an MVA path plasmid;

FIG. 5 illustrates retinoid production and cell growth by a variety ofEscherichia coli strains including pT-DHBSR and pS-NA;

FIG. 6 illustrates retinoid production and cell growth of Escherichiacoli including pT-DHBSR and pS-NA depending on a test volume of aculture solution;

FIG. 7 illustrates retinoid production and cell growth of Escherichiacoli including pT-DHBSR and pS-NA depending on a culture temperature;

FIG. 8 illustrates retinoid production and cell growth of Escherichiacoli including pT-DHBSR and pS-NA depending on a carbon source;

FIGS. 9 and 10 illustrate retinoid production and cell growth ofEscherichia coli including pT-DHBSR and pS-NA depending on aconcentration of glycerol as a carbon source, respectively; and

FIGS. 11 and 12 illustrate results of retinoid production and cellgrowth by a variety of Escherichia coli strains in the presence ofdodecane, respectively.

FIG. 13 illustrates retinoid production and cell growth of Escherichiacoli (pT-DHBSR/pS-NA) depending on a concentration of glycerol as acarbon source in a 2-phase culture system including 1 mL of dodecane in5 ml of culture medium;

FIG. 14 illustrates retinoid production and cell growth of Escherichiacoli (pT-DHBSR/pS-NA) in a 2-phase culture system depending on thevolume of dodecane;

FIG. 15 illustrates distributions of retinoid depending on a culturingtime of Escherichia coli (pT-DHBSR/pS-NA) and a volume of dodecane in a2-phase culture system, which are represented in terms of percentages ofindividual constituents to total retinoid;

FIG. 16 illustrates effects of dodecane addition upon production ofbeta-carotene and cell growth of Escherichia coli including pT-DHB andpS-NA;

FIGS. 17 and 18 illustrate results of retinoid production and cellgrowth of Escherichia coli (pT-DHBSR/pS-NA) in the presence of differentalkanes, respectively;

FIGS. 19, 20 and 21 illustrate results of retinoid production, cellgrowth, and cell specific retinoids productivity of Escherichia coli(pT-DHBSR/pS-NA) in the presence of different volumes of lightweightmineral oil, respectively;

FIGS. 22 and 23 illustrate results of retinoid production and cellgrowth of Escherichia coli (pT-DHBSR/pS-NA) in the presence of heavymineral oil, respectively;

FIGS. 24 and 25 illustrate results of retinoid production and cellgrowth of Escherichia coli (pT-DHBSR/pS-NA) when culturing was conductedby tilting a test tube, respectively;

FIG. 26 illustrates cell growth and pH of Escherichia coli(pT-DHBSR/pS-NA) in the presence of skin-friendly lipophilic substance;and

FIGS. 27 and 28 illustrate results of retinoid production of Escherichiacoli depending on different kinds and amounts of skin-friendlylipophilic substance, respectively.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in more detailsaccording to the following examples. However, these examples areproposed for illustrative purposes only and the scope of the presentinvention is not particularly limited thereto. In the examples, thefollowing experimental materials and methods have been used.

Bacteria Strain and Culture Conditions

Escherichia coli DH5α was used for gene cloning and retinoid production.Alternatively, Escherichia coli MG1655, BL21 (DE3), XL1-Blue, S17-1 andBW25113 were used to investigate an optimum strain for retinolproduction. The culture for retinoid production was executed in a 2YTmedium (including 16 g of tryptone, 10 g of yeast extract and 5 g ofNaCl per liter) at 29° C. using an agitation incubator operating at 250rpm. A major and additional carbon sources were glycerol and arabinose,which were added in concentrations of 0.5 to 2% (w/v) and 0.2% (w/v),respectively, to the incubator. Alternative carbon sources for retinoidproduction, for example, glucose, galactose, xylose and maltose werecompared to glycerol. Ampicillin (100 μg/mL) and chloramphenicol (50μg/mL) were optionally added to a culture solution requiring the same.The culturing was conducted in a test tube containing 7 ml of medium.Cell growth was determined by measuring an optical density at 600 nm(OD₆₀₀). In a 2-phase culture method for production of retinoid, 1 mL ofdodecane (Cat. No. 297879, Sigma, USA) was placed on 5 ml of the culturemedium.

Conditions for Analysis of β-Carotene and Retinoid

β-carotene and retinoid were extracted from bacteria cell pelletsthrough acetone. In the 2-phase culture method including dodecanecapping, cell pieces were completely removed by collecting a dodecanephase containing retinoid and centrifuging the same at 14,000 rpm for 10minutes. The acetone extraction product and dodecane phase were analyzedusing HPLC (LC-20A, Shimadzu, Kyoto, Japan) at detection wavelengths of370 nm (retinal), 340 nm (retinol and retinyl acetate) and 454 nm(β-carotene). The analysis was performed using a Symmetry C18 type (250mm×4.6 mm, 5 m) HPLC column including Sentry Guard C18 (15 mm×4.6 mm, 5m). A mobile phase of the column was each of methanol and acetonitrilein ratios of 95:5 and 70:30, respectively, for analyzing retinoid andβ-carotene. HPLC analysis was performed at a flow rate of 1.5 ml/min anda column temperature of 40° C. Retinal (Cat. No. R2500), retinol (Cat.No. R7632), retinyl acetate (Cat. No. R4632) and β-carotene (Cat. No.C4582) were purchased from Sigma Co. (USA) and dissolved in acetone,respectively, to prepare standard compounds, and each of the preparedstandard compounds was used. Through three independent experiments,results were obtained and represented by a mean±SD.

Example 1 Preparation of Vector for Producing Escherichia coli with HighProductivity of β-Carotene and Retinal

Conventional processes involving genome DNA preparation, restrictionenzyme cleavage, transformation and standard molecular biologicaltechnologies have been executed according to description in relateddocuments (Sambrook and Russell 2001). PCR was performed using pfu DNApolymerase according to standard protocols (Solgent Co., Korea). blhgene of uncultured marine bacteria 66A03 (Genbank accession No.AAY68319) was synthesized into Genofocus (Daejeon, Korea) according tocodon-optimization by Gene Designer software (DNA 2.0, Menlo Park, USA),in order to express the above gene in Escherichia coli.

According to the present example, an enzyme involved in a velocitydetermination process, that is., a gene encoding DXP synthase wasadditionally introduced into Escherichia coli having an MEP path and, atthe same time, a gene encoding an enzyme associated with a mevalonatepath was selected from a variety of gene resources and introduced, thuspreparing Escherichia coli with high productivity of β-carotene.

(1) Preparation of pSNA Vector Including a Gene Encoding an Enzyme in aMevalonate Path Associated with Synthesis of IPP from a Carbon Source

Genes encoding an enzyme in a mevalonate path associated with IPPsynthesis from a carbon source used in the present experiment are shownin Table 1 below.

TABLE 1 Gene sequence (Genbank Name of enzyme Gene accession No.)Acetyl-CoA mvaE SEQ. ID No. 18 acetyltransferase/hydroxymethylglytaryl(AF290092) (HMG)-CoA reductase derived from Enterococcus faecalisHMG-CoA synthase derived from mvaS SEQ. ID No. 19 Enterococcus faecalis(AF290092) Mevalonate kinase derived from mvaK1 SEQ. ID No. 20Streptococcus pneumonia (AF290099) Phosphomevalonate kinase derived frommvaK2 SEQ. ID No. 21 Streptococcus pneumonia (AF290099) Mevalonatediphosphate decarboxylase mvaD SEQ. ID No. 22 derived from Streptococcuspneumoniae (AF290099) Isopentenyl diphosphate (IPP) isomerase Idi SEQ.ID No. 23 derived from Escherichia coli (U00096)

Primers and restriction enzymes to amplify genes listed in Table 1 havebeen described.

TABLE 2 Restriction Primer sequence enzyme mvaE F SEQ. ID No. 37 SacI RSEQ. ID No. 38 SmaI mvaS F SEQ. ID No. 39 SmaI R SEQ. ID No. 40 BamHImvaK1, mvaK2, F SEQ. ID No. 41 KpnI mvaD R SEQ. ID No. 42 XbaI Idi FSEQ. ID No. 43 SmaI R SEQ. ID No. 44 SphI

The primer sequences and restriction enzymes used in cloning the geneslisted in Table 1 are stated in Table 2. Since mvaK1, mvaK2 and mvaD arepresent as a single operon in a chromosome, a whole operon rather thanindividual genes was subjected to PCR cloning at once.

The genes listed in Table 1 were amplified using the primers listed inTable 3 through PCR which uses a chromosome DNA in each strain includingcorresponding gene as a matrix. The amplified product was introducedinto pSTV28 vector (Takara Korea, Korea) (SEQ. ID No. 45) using therestriction enzymes listed in Table 2, thereby preparing the vectorpSNA. The vector pSNA includes all of genes encoding the enzyme in amevalonate path, which can produce IPP from acetyl-CoA.

(2) Preparation of Vectors pT-HB and pT-DHB Including a Gene Encoding anEnzyme Associated with Synthesis of β-Carotene from IPP

Genes encoding an enzyme associated with synthesis of β-carotene fromIPP used in the present experiment, as well as DXP synthase gene as anenzyme involved in the velocity determination process in the MEP path,are shown in Table 3 below.

TABLE 3 Gene sequence (Genbank Name of enzyme Gene accession No.) IPPisophomerase derived from ipiHpl SEQ. ID No. 24 Haematococcus pluvialis(AF082325) 1-deoxyxylolose-5-phosphate (DXP) dxs SEQ. ID No. 25 synthasederived from Escherichia coli (U00096) Geranylgeranyl pyrophosphate(GGPP) crtE SEQ. ID No. 26 synthase derived from pantoea agglomerans(M87280) Phytoene synthase derived from pantoea crtB SEQ. ID No. 27agglomerans (M87280) Phytoene dehydrogenase derived from crtI SEQ. IDNo. 28 pantoea agglomerans (M87280) Lycopene β-cyclase derived frompantoea crtY SEQ. ID No. 29 ananatis (D90087)

TABLE 4 Restriction Gene Primer sequence enzyme ipiHpl F SEQ. ID No. 46SmaISphI R SEQ. ID No. 47 dxs F SEQ. ID No. 48 EcoR1SnaBI R SEQ. ID No.49 crtE F SEQ. ID No. 50 BspHIEcoRI R SEQ. ID No. 51 crtB, crtI F SEQ.ID No. 52 EcoR1SacI R SEQ. ID No. 53 crtY F SEQ. ID No. 54 SalIPstI RSEQ. ID No. 55

The primer sequences and restriction enzymes used in cloning the geneslisted in Table 3 are stated in Table 4. Since crtB and crtI are presentas a single operon in a chromosome, a whole operon rather thanindividual genes was subjected PCR cloning at once.

The genes listed in Table 3 were amplified using the primers listed inTable 4 through PCR which uses a chromosome DNA in each strain includingcorresponding gene as a matrix. The amplified product was introducedinto pTrc99A vector (Genbank accession No. M22744) (SEQ. ID No. 30)using the restriction enzymes listed in Table 4, thereby preparing thevector pT-DHB. The vector pTDHB includes all of genes encoding theenzyme associated with synthesis of β-carotene from IPP, as well as DXPsynthase (dxs) gene as an enzyme used in the velocity determinationprocess in the MEP path. Further, among the genes listed in Table 3, allgenes other than dxs were introduced into pTrc99A vector using therestriction enzymes listed in Table 4, thereby preparing the vectorpT-HB.

(3) Preparation of a Vector Including a Gene Encoding an EnzymeAssociated with Synthesis of Retinal from β-Carotene

Genes encoding an enzyme associated with synthesis of retinal fromβ-carotene used in the present experiment are shown in Table 5 below. Asa gene encoding β-carotene monooxygenase derived from uncultured marinebacterium 66A03, SR gene which is an Escherichia coli codon-optimizedsequence of blh was used.

TABLE 5 Gene sequence (Genbank Name of enzyme Gene accession No.)β-carotene monooxygenase derived blh SEQ. ID No. 31 from unculturedmarine bacterium (DQ065755) 66A03 β-carotene monooxygenase derived SRSEQ. ID No. 32 from uncultured marine bacterium (Escherichia 66A03 colicodon- optimized sequence of blh) β-carotene 15,15′-monooxygenase BcmoISEQ. ID No. 33 derived from Mus musculus (NM_021486) Brp-like protein 2derived from brp2 SEQ. ID No. 34 Natronomonas pharaonis ATCC35678(CR936257) β-carotene monooxygenase derived Blh SEQ. ID No. 35 fromHalobacterium salinarum (AE004437) ATCC700922 β-carotene monooxygenasederived Brp SEQ. ID No. 36 from Halobacterium salinarum (AE004437)ATCC700922

TABLE 6 Restriction Gene Primer sequence enzyme SR F SEQ. ID No. 56EcoR1SpeI R SEQ. ID No. 57 bcmo1 F SEQ. ID No. 58 EcoR1SpeI R SEQ. IDNo. 59 brp2 F SEQ. ID No. 60 EcoR1SpeI R SEQ. ID No. 61 blh F SEQ. IDNo. 62 EcoR1SpeI R SEQ. ID No. 63 brp F SEQ. ID No. 64 EcoR1SpeI R SEQ.ID No. 65

The primer sequences and restriction enzymes used in cloning the geneslisted in Table 5 are stated in Table 6. The genes listed in Table 5were amplified using the primers listed in Table 6 through PCR whichuses a chromosome DNA in each strain including a corresponding gene as amatrix. The amplified product was introduced into pT-HB vector using therestriction enzymes listed in Table 6, respectively, thereby preparingthe vectors pT-HBSR, pT-HBBcmo1, pT-HBbrp2, pT-HBblh and pT-HBbrp. Suchvectors pT-HBSR, pT-HBBcmo1, pT-HBbrp2, pT-HBblh and pT-HBbrp arevectors formed by introducing SR, Bcmo1, brp2, blh and brp genes intopT-HB vector, respectively, and have included all of genes encoding anenzyme associated with the synthesis of retinal through β-carotene fromIPP. After cutting SR gene from pT-HBSR using SpeI, the cut gene wasintroduced into a corresponding part of pT-DHB, thus preparing pT-DHBSR.

Example 2 Comparison of Different BCM(D)O Genes in Relation to RetinalProduction

Retinal may be produced by introduction of BCM(D)O gene encodingβ-carotene mono(di)oxygenase, which is a recombinant Escherichia coliproducing β-carotene. The present inventors have conducted cloning ofBCM(D)O gene from two bacteria, i.e., Halobacterium sp NRC-1 (blh andbrp genes) and Natronomonas pharaonis (brp2 gene), as well as Musmusculus (Bcmo1 gene) of a vertebrate animal. The present inventors havesynthesized codon-optimized BCDO gene (SR) on the basis of an amino acidsequence of uncultured marine bacterium 66A03 blh gene. BCM(D)O gene(SR) was used to prepare retinal synthetic plasmids pT-HBblh, pT-HBbrp,pT-HBbrp2, pT-HBBcmo1 and pT-HBSR, respectively. The recombinantEscherichia coli cell containing each of retinal plasmids was culturedin a 2YT medium including 0.5% (w/v) of glycerol and 0.2% (w/v) ofarabinose as a carbon source at 29° C. for 48 hours.

FIG. 3 illustrates production of retinal, production of β-carotene andcell growth of Escherichia coli including pT-HB, pT-HBblh, pT-HBbrp,pT-HBbrp2, pT-HBBCMO1 and pT-HBSR. More particularly, white and greybars show numerical values at 24 hours and 48 hours, respectively.

As shown in FIG. 3, the recombinant Escherichia coli pT-HBblh, pT-HBbrpand pT-HBSR have produced 2.2, 0.8 or 1.4 mg/L of retinal, respectively,at 24 hours. However, retinal production by the recombinant Escherichiacoli pT-HBblh or pT-HBbrp was reduced to 0.7 or 0.4 mg/L, respectively,at 48 hours, whereas Escherichia coli pT-HBSR showed a slight increasein retinal production. The decrease in retinal production after 24 hoursmay be caused by oxidative degradation of retinal in the cell. An amountof retinal obtained from the culture solution depends upon bothintracellular synthesis and degradation of retinal.

For Escherichia coli including pT-HBblh or pT-HBbrp, a retinalproductivity at 24 hours after culturing may be lower than a rate ofdegradation of the same. In the culture of Escherichia coli strainincluding pT-HBbrp2 or pT-HBBcmo1, a trace amount of retinal wasdetected. Escherichia coli without BCM(D)O gene has produced 35 mg/L ofβ-carotene, but did not produce retinal. Since β-carotene is a precursorjust before retinal, a β-carotene consumption by BCM(D)O may be exactlyproportional to the retinal productivity if there was retinaldegradation. β-carotene remained in a culture solution of Escherichiacoli including BCM(D)O other than SR, therefore, β-carotene cleavageactivity of SR was expected to be the highest level among testedBCM(D)O. Accordingly, in an additional experiment, SR enzyme was adoptedfor retinal production. The cell growth did not come under the influenceof over-expression of BCM(D)O gene except for N. pharaonis brp geneexhibiting delayed cell growth.

Example 3 Gene Manipulation into MEP and MVA Paths for Supplying aBuilding Block

Retinal building blocks, that is, IPP and DMAPP may be synthesized inEscherichia coli through an inherent MEP path and a foreign MVA path(FIG. 1).

It was reported that synthesis of 1-deoxy-d-xylolose-5-phosphate (DXP)is an important velocity restriction process in the MEP path. Therefore,over-expression of DXP synthase (to be encoded by dxs) increasedproduction of lycopene and β-carotene in previous inventions of thepresent inventors. By introducing dxs gene into before the MEP pathamong pT-HBSR, pT-DHBSR was prepared.

FIG. 4 illustrates production of retinal and β-carotene, and cell growthof Escherichia coli including pT-HB, pT-HBSR, pT-DHB and pT-DHBSR, aswell as Escherichia coli including pT-DHB or pT-DHBSR together withpS-NA as an MVA path plasmid. More particularly, white and grey barsshow numerical values at 24 hours and 48 hours, respectively.

As shown in FIG. 4, the retinal productivity of Escherichia colipT-DHBSR was a little higher than that of Escherichia coli pT-DHB at 24hours, while being substantially similar to the same at 48 hours.However, β-carotene production by Escherichia coli pT-DHB was increasedby about 1.5 times due to over-expression of dxs, as compared toEscherichia coli pT-HB. It is known that a foreign MVA path inEscherichia coli considerably increases production of isoprenoid byproviding sufficient amounts of IPP and DMAPP building blocks.Escherichia coli pT-DHBSR/pS-NA including an additional foreign MVA pathhave produced 8.7 mg/L of retinal for 48 hours, which is 4 times higherthan the productivity of Escherichia coli pT-DHBSR. For an Escherichiacoli strain including SR gene, β-carotene did not remain or slightlyremained in the cell. This condition is presumed due to an effectivecleavage reaction of β-carotene by SR. There was a considerabledifference between an amount of β-carotene (a substrate) consumption andan amount of produced retinal (a product). This difference may be due tothe presence of a cellular reaction to metabolize retinal in Escherichiacoli as well as biological degradation of retinal. Accordingly,formation of an alternative retinoid derived from retinal by any enzymein Escherichia coli may be under consideration. Since the retinal can beconverted into retinol, retinoic acid and retinyl ester by cell-enzymereaction (FIG. 2), retinal derivatives contained in an Escherichia coliculture solution were subjected to analysis. The analysis results showedthat retinal derivatives other than retinoic acid were formed. Accordingto further experiments, production of retinal, retinol and retinylacetate was determined.

Example 4 Effects of Escherichia coli Strain, Culture Conditions andCarbon Source in Relation to Retinoid Production

(1) Strain

With regard to production of retinoid including retinal, retinol andretinyl acetate, effects of Escherichia coli strains were investigated.Five Escherichia coli strains including pT-DHBSR and pS-NA, that is,MG1655, DH5α, XL1-Blue, S17-1 and BL21 (DE3) were used to produceretinoid. Table 7 shows characteristics of six Escherichia coli strainsincluding the foregoing five strains.

TABLE 7 E. coli strain Details MG1655 K12, wild type DH5α F⁻,φf80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1endA1, hsdR17(r_(K) ⁻m_(K)⁺), phoA, supE44, λ−, thi-1, gyrA96, relA1 XL1-Blue hsdR17, supE44,recA1, endA1, gyrA46, thi, relA1, lac/F′[proAB⁺, lacI^(q),lacZΔM15::Tn10(tet^(r))] S17-1 recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 BL21(DE3) F⁻, ompT, hsdS_(B)(r_(B) ⁻m_(B) ⁻), gal(lcI857, ind1, Sam7, nin5,lacUV5-T7 gene1), dcm(DE3) BW25113 Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),lambda⁻, rph-1, Δ(rhaD-rhaB)568, hsdR514

FIG. 5 illustrates retinoid production and cell growth of fiveEscherichia coli strains, respectively, of which each has pT-DHBSR andpS-NA. Culturing was conducted in a 2YT medium including 0.5% (w/v) ofglycerol and 0.2% (w/v) of arabinose at 29° C. for 48 hours. Retinal,retinol and retinyl acetate are represented by bright grey, dark greyand black colors, respectively. Also, in case of cell growth, MG1655,DH5α, XL1-Blue, S17-1 and BL21 (DE3) are represented by ▪, , ▴, ▾ and♦, respectively.

As shown in FIG. 5, Escherichia coli DH5α has produced 40 mg/L, that is,the largest amount of retinoid at 36 hours, and Escherichia coli S17-1and XL1-Blue have produced about 22 mg/L, that is, the second largestamount of retinoid. However, from Escherichia coli MG1655 and BL21(DE3), only a trace amount of retinoid was produced. Therefore,Escherichia coli DH5α was adopted as a strain for retinoid production.

(2) Culture Conditions

With regard to production of retinoid, effects of dissolved oxygen wereinvestigated with difference test volumes in a test tube having adiameter of 30 mm.

FIG. 6 illustrates retinoid production and cell growth of Escherichiacoli including pT-DHBSR and pS-NA depending on test volume. Referring toFIG. 6, retinal, retinol and retinyl acetate are represented by brightgrey, dark grey and black colors, respectively. Also, in case of cellgrowth, test volumes of 3 mL, 5 mL, 7 mL and 10 mL are represented by ▪,▪, ▴ and ▾, respectively. Culturing was conducted in a 2YT mediumincluding 0.5% (w/v) of glycerol and 0.2% (w/v) of arabinose at 29° C.for 48 hours.

As shown in FIG. 6, it was found that retinoid production more earlyreached the maximum level at a small test volume (corresponding tohighly dissolved oxygen), and the production was deemed to more quicklydecrease due to oxidative degradation. With 10 mL of test volume, bothof cell growth and retinoid production were delayed, while degradationof the product was observed by a small extent. It was found that theoptimum test volume for retinoid production is 7 mL.

Further, the retinoid production depending upon the temperature wasinvestigated. FIG. 7 illustrates retinoid production and cell growth ofEscherichia coli including pT-DHBSR and pS-NA depending on a culturetemperature. Referring to FIG. 7, retinal, retinol and retinyl acetateare represented by bright grey, dark grey and black colors,respectively. Also, in case of cell growth, culture temperatures of 29°C., 34° C. and 37° C. are represented by ▪,  and, respectively.Culturing was conducted in a 2YT medium including 0.5% (w/v) of glyceroland 0.2% (w/v) of arabinose at 29° C., 34° C. and 37° C., respectively,for 48 hours.

As shown in FIG. 7, the retinoid production has come under an influenceof culture temperature and the highest production was accomplished at29° C.

(3) Carbon Source

Effects of different carbon sources upon production of retinoid werecompared.

FIG. 8 illustrates retinoid production and cell growth of Escherichiacoli including pT-DHBSR and pS-NA depending on the carbon source.Referring to FIG. 8, retinal, retinol and retinyl acetate arerepresented by bright grey, dark grey and black colors, respectively.Also, in case of cell growth, no carbon source, and the carbon sourcesof glycerol, glucose, xylose, maltose and galactose are represented by▪, , ▴, ▾, ♦ and, respectively. Culturing was conducted in a 2YT mediumincluding 0.2% (w/v) of arabinose and 0.5% (w/v) of glycerol, glucose,xylose, maltose or galactose, at 29° C. for 48 hours.

As shown in FIG. 8, it was found that glycerol was the best carbonsource for retinoid production. When glucose or galactose was used asthe carbon source, the retinoid productivity was lower than that in casewhere the carbon source was not used.

Next, effects of a concentration of glycerol upon the retinoidproduction and cell growth were investigated. Escherichia coliDH5α(pT-DHBSR/pSNA) was grown in a 2YT medium including glycerol in arange of 0.0% to 2.0% (w/v), at 29° C.

FIG. 9 illustrates production of retinoids (retinal, retinol and retinylacetate) of Escherichia coli including pT-DHBSR and pS-NA. Retinal,retinol and retinyl acetate are represented by bright grey, dark greyand black colors, respectively.

FIG. 10 illustrates cell growth of Escherichia coli including pT-DHBSRand pS-NA. Given glycerol concentrations of 0%, 0.5%, 1% and 2% arerepresented by ▪, , ▴ and ▾, respectively.

As shown in FIGS. 9 and 10, the cell growth was proportional to theglycerol concentration and increased. With the glycerol concentration of0.5, 1.0 and 2.0% (w/v), the cell growth has become stagnate at 36, 48and 72 hours, respectively. At the above time, the maximum retinoidproductivity was accomplished and, thereafter, the productivity wasconsiderably reduced during stagnation. It can be seen that the retinoidproduction generally increases after 24 hours. The retinoid productivitywas about 95 mg/L, the highest level, at 2.0% (w/v) of glycerol amongvarious glycerol concentrations, which is substantially 2.4 times higherthan the maximum retinoid productivity at 0.5% (w/v) of glycerol. Anincrease in glycerol concentration delayed the stagnation whileextending a period of retinoid production.

From all culture solutions, it was observed that the retinoid productionwas extremely reduced during stagnation of the cell growth, and thiscondition is deemed to be caused by discontinued production of retinoidduring stagnation and intracellular oxidative degradation of the same.

(4) Culture in the Presence of Dodecane

Strains containing transformed pT-DHBSR/pSNA were used for six strainslisted in Table 7. After adding 1 mL of dodecane to 5 ml of medium,culturing was conducted according to such conditions as described in“bacteria strain and culture conditions.” The medium used herein was a2YT medium including 0.2% (w/v) of arabinose and 0.5% (w/v) of glyceroladded thereto.

FIG. 11 illustrates results of the retinoid production depending ondifferent Escherichia coli strains for retinoid production. As shown inFIG. 11, DH5α and MG1655 showed the largest amount of retinoidproduction. For MG1655, the cell growth and retinoid productivity wereincreased, as compared to no addition of dodecane. A cell growth rateand a rate of increasing retinoid productivity for MG1655 were obviouslyfaster than DH5α. BL21 (DE3) strain showed a still high cell growth buthad scarcely any production of retinyl acetate. Consequently, it wasdetermined that DH5α and MG1655 among six strains are relativelysuitable, as compared to other strains.

FIG. 12 illustrates results of growth of strains for retinoid productionin the presence of dodecane.

FIG. 13 illustrates results of retinoid production and growth in thepresence of dodecane, depending on the concentration of glycerol as acarbon source.

Example 5 2-Phase Culture Using Dodecane for In-Situ Extraction ofRetinoid

In order to prevent intracellular degradation of retinoid, a 2-phaseculture method was conducted using a hydrophobic solvent, that is,dodecane, in order to perform in-situ extraction of retinoid from cells.Dodecane was selected since it has a low toxicity to Escherichia coli I,a high hydrophobicity (log P_(o/w), 6.6) for extracting hydrophobicretinoid, and low volatile properties not to cause evaporation loss.

In the present example, 1 mL of dodecane was added to 5 ml of culturesolution. FIG. 13 illustrates retinoid production and cell growth ofEscherichia coli (pT-DHBSR/pS-NA) in a 2-phase culture system including1 mL of dodecane in 5 ml of culture medium. With regard to retinoidproduction, retinal, retinol and retinyl acetate are represented bybright grey, dark grey and black colors, respectively. In the case ofcell growth, given glycerol concentrations of 0.5%, 1% and 2% arerepresented by ▪,  and ▴, respectively.

Retinoid was extracted into the dodecane phase while an insignificantamount of retinoid was detected in the culture solution and cell mass(data not shown). As a result, the retinoid productivity was measuredfrom the dodecane phase. As shown in FIG. 13, in-situ extraction couldminimize intracellular degradation of retinoid by dodecane. The retinoidamong the dodecane phase was deemed to be relatively stable and remainedwithout significant oxidative degradation thereof. As comparing with theresults shown in FIGS. 9 and 10 (without addition of dodecane), theretinoid production was remarkably increased even at 24 hours in case ofadding the dodecane. Further, the cell growth did not come under aninfluence of the dodecane addition while the retinoid production was notdecreased during stagnation. However, in the culture using 2% (w/v)glycerol, the retinoid production was not so higher than that in case ofusing 1% (w/v) glycerol, even though the cell growth was remarkablyincreased in proportion to an increase in glycerol concentration from 1%(w/v) to 2% (w/v). When the volume of dodecane addition is 1 mL, it isinsufficient to conduct effective in-situ extraction of retinoid in theculture using 2% (w/v) of glycerol.

In order to investigate effects of the volume of dodecane addition onthe retinoid production and cell growth, 1 mL to 5 mL of dodecane wasinitially added to a culture solution including 2% (w/v) of glycerol(FIG. 14).

FIG. 14 illustrates retinoid production and cell growth of Escherichiacoli (pT-DHBSR/pS-NA) in a 2-phase culture system, depending on thevolume of dodecane. With regard to the retinoid production, retinal,retinol and retinyl acetate are represented by bright grey, dark greyand black colors, respectively. In case of cell growth, volumes ofoverlaying dodecane of 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL arerepresented by ▪, , ▴, □, ∘, Δ and ⋆, respectively.

FIG. 15 illustrates a distribution of retinoid depending on culturingtime and volume of dodecane, in terms of percentages of individualconstituents to total retinoid. Retinal, retinol and retinyl acetate arerepresented by bright grey, dark grey and black colors, respectively.

As shown in FIGS. 14 and 15, production of overall retinoids wasimproved according to an increase in the volume of dodecane addition. In72 hours culture using 5 mL of dodecane, the highest retinoidproductivity of 136 mg/L was obtained, which is about 2 times highervalue than that (65 mg/L) in case where 1 mL of dodecane is used.Meanwhile, in 72 hours-extended culture using 5 mL of dodecane, theretinoid productivity did not further increase but the highest level wasmaintained without degradation of the retinoid (data not shown). Byadding 2 mL of dodecane to the culture solution at 0, 24 and 48 hours, awhole volume of dodecane addition was increased to 6 mL. In the cultureusing 6 mL of added dodecane, the total retinoid productivity did notincrease, as compared to the culture using 5 mL of dodecane. Likewise,even in the culture using 6 mL of initially added dodecane, the retinoidproductivity did not increase (data not shown). The cell growth in allof culture solutions including dodecane was slightly higher than that incase of not using dodecane (FIG. 14).

FIG. 15 illustrates a distribution of produced retinoids depending onthe volume of dodecane addition. With regard to ratios of obtainedretinal and retinol, there is a considerable difference in retinoiddistributions between addition of dodecane and no addition of dodecane.A ratio of retinal in retinoid at 48 hours was about 51% (w/v) in thedodecane-added culture and 23% (w/v) in the culture without addingdodecane. Likewise, a ratio of retinol ranged from 30 to 39% in thedodecane-added culture and was 59% in the culture without addingdodecane. Accordingly, the addition of dodecane may increase a ratio ofretinal while reducing a ratio of retinol. In consideration of the orderof reactions for formation of retinol from retinal in a cell, retinal isdeemed to be extracted from the cell before conversion of the same intoretinol by dodecane. Further, a ratio of retinyl acetate at 48 hours wasless than 20% in both of the cultures with and without addition ofdodecane, which is relatively lower than the ratios of retinal andretinol. In the culture with addition of dodecane, the ratio of retinylacetate is reduced as the culturing time is extended and this indicatesthat activity of cells for forming retinyl acetate is reduced duringculturing. Consequently, adding dodecane has prevented a decrease ofretinoid production during stagnation of the cell growth, whileimproving the retinoid production.

The in-situ extraction of retinoid according to the present inventiondoes not need lysozyme used for degrading a cell wall. Retinoid (C20,isoprenoid molecule) may be efficiently released from the cell withoutloss of the cell wall. In 2-phase culture for production of retinoid,β-carotene must be continuously maintained in the cell since it is adirect precursor of the retinoid. If β-carotene is extracted from thedodecane phase, it can be cut by BCD(M)O placed in cytosol.

Due to a size of molecule, β-carotene can neither be released from thecell nor extracted by dodecane, therefore, can be continuouslymaintained in the cell during 2-phase culture of β-carotene (FIG. 16).

FIG. 16 illustrates effects of dodecane addition depending on β-caroteneproduction and cell growth of Escherichia coli including pT-DHB andpS-NA. Culturing was conducted in 5 ml of 2YT medium including 0.5%(w/v) of glycerol and 0.2% (w/v) of arabinose at 29° C. for 48 hourswhile adding 1 mL of dodecane to the medium. Grey and black barsindicated 24 hours and 48 hours, respectively.

As shown in FIG. 16, an insignificant amount of β-carotene was detectedin the dodecane phase and a whole β-carotene has been almost retained inthe cell. There was not a noticeable difference in β-carotene productionand cell growth between cultures with and without addition of dodecane.

In the culture with addition of 5 mL of dodecane, a total 122 mg/L ofretinoid productivity was attained at 48 hours. However, in the culturewithout addition of dodecane, only half of the above productivity (60mg/L) was obtained at the same time period. Accordingly, thedodecane-added 2-phase culture system may be appropriately applied to analternative transformation system to produce small lipophilic molecules.

Example 6 Production of Retinoid in Medium Including LipophilicSubstance

The present example was performed to identify as to whether a variety oflipophilic substances have effects of increasing retinoid production.

(1) Production of Retinoid in Medium Including Alkane

A strain DH5α including transformed pT-DHBSR/pSNA (DH5α(pT-DHBSR/pSNA))was used, and after adding 5 mL of each of octane, decane, dodecane andtetradecane to 5 ml of medium, culturing was conducted according to suchconditions as described in “Bacteria strain and culture conditions.” Themedium used herein was a 2YT medium including 0.2% (w/v) of arabinoseand 2.0% (w/v) of glycerol added thereto.

FIG. 17 illustrates results of retinoid production in the presence ofalkane. FIG. 18 illustrates results of growth of the retinoid producingstrain in the presence of alkane.

As shown in FIG. 17, a total 108 mg/L of retinoid was produced in caseof using decane. Alternatively, bacterial cell proliferation, pH and anamount of β-carotene in the bacterial cell did not show a considerabledifference depending upon the presence of alkanes. Therefore, it isconsidered that decane may be more advantageous in retinoid production,as compared to dodecane. When using octane, production of retinal andretinol was similar to other alkanes, whereas retinyl acetate was almostnot produced. Tetradecane showed a lower productivity of wholeretinoids, as compared to other alkanes.

(2) Production of Retinoid in Medium Including Mineral Oil

(2.1) Lightweight Mineral Oil

The lightweight mineral oil is cheap and has an economical advantage, ascompared to alkanes. A strain DH5α including transformed pT-DHBSR/pSNA(DH5α(pT-DHBSR/pSNA)) was used, and after adding the lightweight mineraloil in different volumes to 5 ml of medium, respectively, culturing wasconducted according to such conditions as described in “Bacteria strainand culture conditions.” The medium used herein was a 2YT mediumincluding 0.2% (w/v) of arabinose and 2.0% (w/v) of glycerol addedthereto.

FIG. 19 illustrates results of retinoid production in the presence oflightweight mineral oil. FIG. 20 illustrates results of strain growth inthe presence of lightweight mineral oil.

As shown in FIG. 19, 158 mg/L of retinoid was produced in the presenceof the lightweight mineral oil in an amount of 2 ml, as compared to136.1 mg/L of retinoid produced using 5 mL of dodecane. As shown in FIG.20, pH was not considerably different other than the case of usingdodecane. On the other hand, bacterial cell growth was reduced as anamount of the lightweight mineral oil increased. The reason of thiscondition was deemed because the medium and mineral oil were notsufficiently admixed due to a high viscosity and specific gravity of thelightweight mineral oil. Owing to a decrease in growth of the bacterialcell, the retinoid production was also reduced.

FIG. 21 illustrates cell specific retinoids productivity. As shown inFIG. 21, a specific productivity of about 5 mg/L/OD₆₀₀ nm was observedregardless of an amount of mineral oil.

(2.2) Heavy Mineral Oil

The heavy mineral oil is cheaper than the lightweight mineral oil. Astrain DH5α including transformed pT-DHBSR/pSNA (DH5α(pT-DHBSR/pSNA))was used, and after adding 2 ml of heavy mineral oil to 5 ml of medium,culturing was conducted according to such conditions as described in“Bacteria strain and culture conditions.” The medium used herein was a2YT medium including 0.2% (w/v) of arabinose and 2.0% (w/v) of glyceroladded thereto.

FIG. 22 illustrates results of retinoid production in the presence ofheavy mineral oil. FIG. 23 illustrates results of strain growth in thepresence of heavy mineral oil. As shown in FIGS. 22 and 23, the heavymineral oil involved lower cell growth, as compared to the lightweightmineral oil and dodecane. Further, 104.6 mg/L of retinoid was produced.The reason of this condition was deemed because the medium and mineraloil were not sufficiently admixed due to a viscosity of the heavymineral oil.

Except that a test tube was tilted and mounted on an incubator, cellculture was performed by the same procedures as described above. Bytilting the test tube, effects of agitation were improved to thus allowthe medium and mineral oil to be admixed more effectively.

FIG. 24 illustrates results of retinoid production when the culturingwas conducted in a tilted test tube. FIG. 25 illustrates results ofstrain growth when the culturing was conducted in the tilted test tube.As shown in FIGS. 24 and 25, the cell growth and retinoid productionwere increased when the culturing was conducted in the tilted test tube.More particularly, the retinoid was produced in an amount of 88.2 mg/Lat 96 hours in a vertically-mounted test tube, while the retinoidproductivity reached 173.9 mg/L in the tilted test tube.

The above results indicated that mixing the lightweight and/or heavymineral oils with the medium is an important factor in retinoidproduction since the mineral oils have a high viscosity. Accordingly,the foregoing lightweight and/or heavy mineral oils may be used forretinoid by properly agitating the same during culturing.

(3) Production of Retinoid in Medium Including Skin-Friendly LipophilicSubstance

Retinoid was produced in a medium including a skin-friendly lipophilicsubstance. As the skin-friendly lipophilic substance, isopropylmyristate (IPM), dioctanoyl-decanoyl glycerol (ODO), cetylethylhexanoate (CEH) and phytosqualane were used.

A strain DH5α including transformed pT-DHBSR/pSNA (DH5α(pT-DHBSR/pSNA))was used, and after adding 2 ml of heavy mineral oil to 5 ml of medium,culturing was conducted according to such conditions as described in“Bacteria strain and culture conditions.” The medium used herein was a2YT medium including 0.2% (w/v) of arabinose and 2.0% (w/v) of glyceroladded thereto. A control was prepared by adding 5 mL of dodecane to themedium.

FIG. 26 illustrates cell growth and pH in the presence of skin-friendlylipophilic substance. FIGS. 27 and 28 illustrate results of retinoidproduction depending on an amount of skin-friendly lipophilic substance.As shown in FIGS. 27 and 28, in case of lipophilic substances other thandodecane, using 2 ml of lipophilic substance has achieved an increase inretinoid productivity, as compared to 5 ml of the same. In other words,when 2 ml of lipophilic substance was added to 5 ml of medium includingthe lightweight mineral oil, IPM, ODO, CEH and phytosqualane, largeamount of retinoid was produced. Especially, when using IPM among IPM,ODO, CEH and phytosqualane, the largest amount of retinoid was produced.More specifically, when adding 2 ml of IPM, 180 mg/L of retinoid wasproduced. For IPM, in consideration of similar growth of bacterial cell,it is presumed to have a high specific productivity per bacterial cell.

1. A method for production of retinoid from a microorganism, comprising:culturing a microorganism having retinoid producing efficacy in a mediumcontaining a lipophilic substance; and isolating the retinoid from thelipophilic substance.
 2. The method according to claim 1, wherein themicroorganism is bacteria, fungi, isolated animal cell or a combinationthereof.
 3. The method according to claim 1, wherein the microorganismis the genus Escherichia, the genus bacillus, the genus corynebacterium,yeast, kluyveromyces or a combination thereof.
 4. The method accordingto claim 1, wherein the lipophilic substance is an alkane compoundhaving 8 to 50 carbon atoms, a compound represented by Formula 1 below,a compound represented by Formula 2 below, or a combination thereof:R₁(CO)OR₂  [Formula 1] wherein R₁ and R₂ are each independently alkylhaving 8 to 50 carbon atoms, and CO represents a carbonyl group; and

wherein R₃, R₄ and R₅ are each independently alkyl having 8 to 50 carbonatoms, and CO represents a carbonyl group.
 5. The method according toclaim 1, wherein the lipophilic substance is octane, decane, dodecane,tetradecane, phytosqualane, mineral oil, isopropyl myristate, cetylethylhexanoate, dioctanoyl decanoyl glycerol, squalane, or a combinationthereof.
 6. The method according to claim 1, wherein a ratio by volumeof the medium to the lipophilic substance ranges from 1:0.2 to 3.0. 7.The method according to claim 1, wherein the culturing is performedwhile agitating.
 8. The method according to claim 1, wherein the mediumfurther includes glycerol.
 9. The method according to claim 1, whereinthe medium further includes glucose.
 10. The method according to claim1, wherein the isolating includes removing cells from a culture solutionand then isolating the retinoid from dodecane.
 11. The method accordingto claim 1, wherein the retinoid is at least one selected from a groupconsisting of retinal, retinol, retinyl ester and retinoic acid.
 12. Themethod according to claim 1, wherein the microorganism is Escherichiacoli.
 13. The method according to claim 12, wherein the Escherichia coliis DH5α, MG1655, BL21 (DE), S17-1, XL1-Blue, BW25113 or a combinationthereof.
 14. The method according to claim 1, wherein the microorganismis one transformed into: a gene encoding acetyl-CoA acetyltransferase/hydroxymethylglutaryl(HMG)-CoA reductase derived fromEnterococcus faecalis, which is defined by SEQ. ID No. 1; a geneencoding HMG-CoA synthase derived from Enterococcus faecalis, which isdefined by SEQ. ID No. 2; a gene encoding mevalonate kinase derived fromStreptococcus pneumoniae, which is defined by SEQ. ID No. 3; a geneencoding phosphomevalonate kinase derived from Streptococcus pneumoniae,which is defined by SEQ. ID No. 4; a gene encoding mevalonatediphosphate decarboxylase derived from Streptococcus pneumoniae, whichis defined by SEQ. ID No. 5; a gene encoding isopentinyl diphosphate(IPP) isomerase derived from Escherichia coli, which is defined by SEQ.ID No. 6; a gene encoding geranylgeranyl pyrophosphate (GGPP) synthasederived from Pantoea agglomerans, which is defined by SEQ. ID No. 7; agene encoding phytoene synthase derived from Pantoea agglomerans, whichis defined by SEQ. ID No. 8; a gene encoding phytoene dehydrogenasederived from Pantoea agglomerans, which is defined by SEQ. ID No. 9; anda gene encoding lycopene β-cyclase derived from Pantoea ananatis, whichis defined by SEQ. ID No.
 10. 15. The method according to claim 14,wherein the microorganism is one further transformed into at least oneselected from a group consisting of: a gene encoding β-carotenemonooxygenase derived from uncultured marine bacterium 66A03, which isdefined by SEQ. ID No. 13; a gene encoding β-carotene15,15′-monooxygenase derived from Mus musculus, which is defined by SEQ.ID No. 14; a gene encoding brp-like protein 2 (brp 2) derived fromNatronomonas pharaonis ATCC35678, which is defined by SEQ. ID No. 15;and a gene encoding β-carotene monooxygenase derived from Halobacteriumsalinarum ATCC700922, which is defined by SEQ. ID No. 16 or
 17. 16. Themethod according to claim 14, wherein the microorganism is one furthertransformed into a gene having a base sequence defined by SEQ. ID No.32, which is codon-optimized in Escherichia coli.
 17. The methodaccording to claim 1, wherein the microorganism is one transformed intoa gene encoding 1-deoxyxylolose-5-phosphate (DXP) synthase derived fromEscherichia coli, which is defined by SEQ. ID No.
 11. 18. The methodaccording to claim 1, wherein the microorganism is one transformed intoa gene encoding IPP isomerase derived from Haematococcus pluvialis,which is defined by SEQ. ID No.
 12. 19. The method according to claim 1,wherein the microorganism in the genus Escherichia is Escherichia coliDH5α/pTDHB/pSNA which is deposited under Accession No. KCTC 11254BP orEscherichia coli DH5α/pTDHBSR/pSNA which is deposited under AccessionNo. KCTC 11255BP.